Fructooligosaccharides are sugars found in nature, which, when consumed, provide several benefits to the health of a person.
GF2 (1-kestose), GF3 (nystose) and GF4 (fructofuranosil nystose) are composed of glucose units, to which are bound one, two or even four fructose units.
These sugars can be found in a large quantity of foods provided by nature, such as asparagus, banana, garlic, onion, tomato or wheat. Besides giving a sweetish flavor to foods, they are neither cariogenic nor caloric and promote the population growth of the bifidus bacteria in the intestine, which reduce the activity of the putrefactive bacteria, thereby reducing the development of toxic products by fermentation.
In view of the benefits described above, the interest for fructooligosaccharides have raised progressively. Consequently, intensive investigations are now being undertaken with the aim of obtaining these fructooligosaccharides from enzymes. It is known that the beta-fructofuranosidase enzyme, which is used in the production of fructooligosaccharides by transfer activity can be obtained in different ways, particularly from the cultures of fungi of different species, such as Aspergillus, Pennicillium, Fusarium, Gloesporium, from the cultures of yeasts, such as Saccharomyces, Rhodotorulla, Pichia, Hansenula, Candida and Aureobasidium, and also from some plants, such as asparagus. It is also known that this enzyme may be prepared in different ways and under different process conditions. Said enzyme is also known for promoting the catalyzation of the transfructosylation reaction, which is responsible for transferring the fructosyl group from a donor to a receptor, which may be sucrose or a fructooligosaccharide, such as kestose, nystose, etc. Nevertheless, the structure of this enzyme is still unknown.
The manner by which the transfructosylation of the Aspergillus orizac occurs has been studied before, regarding its performance in the formation of fructose oligomers, by using sucrose as raw material.
It has been observed that the hydrolysis of sucrose with extracts of Pennicillium spinulosum was initially fast and followed by the formation of non-reducing fructooligosaccharides. The hydrolysis of these fructooligosaccharides occurred by transferring the fructose units to the water and eventually resulted in the complete hydrolysis of the sucrose. The transfructosylation and inversion activities occurred from the use of the same enzyme, i.e., the beta-fructofuranosidase (Bealing et al., Biochem J 53(2):277–285 (1953); Bealing, Biochem J 55(1):93–101 (1953)). From U.S. Pat. No. 4,849,356 fructooligosaccharides were produced with mycelium extracts, by culturing the fungus Aspergillus phoenics in an adequate culture medium. According to this document, the enzyme is preferably prepared on a solid substrate mostly containing sucrose. The beta-fructofuranosidase enzyme thus obtained is cell-bound and requires, to be recovered, complex operations for separating the mycelium from the liquid phase. In this prior art process, in order to obtain a good yield, the presence of sucrose is still required in the culture medium. Moreover, the enzyme obtained as described above provides only the formation of lower fructose oligomers, such as GF2, GF3, with the production of GF4 not being demonstrated.
The formation of fructooligosaccharides has also been investigated, by using cell suspension of several other fungi. Among these fungi, the Aspergilus niger ATCC 20611 produced the highest level of the activity of the beta-fructofuranosidase enzyme, as compared to the activity of hydrolysis (Hidaka et al, Agric Biol Chem, 52(5):1181–1187 (1988)). The enzyme was subsequently purified and then characterized.